The completion of the human genome sequence analysis is prompting a trend towards applying genetic information to diagnoses and the like in a medical field. A typical DNA analysis technique is gel electrophoresis and particularly capillary array gel electrophoresis apparatus is widely used as a high-speed, high throughput DNA analytical system and also for genome analysis. Following the genome sequence analysis, noticeable are gene expression profile analysis and analysis of single nucleotide polymorphisms (SNPs) in genes. Functions of genes and correlations between genes and diseases or drug sensitivity have been studied by examining genes expressed under various conditions or various gene mutations in individuals. Further, diagnosis of diseases and the like using such accumulated information on genes are about to be implemented.
In diagnoses of diseases, unlike in analysis of unknown gene, subjects to be tested are known genes or the presence or absence of mutations in such genes. The tests are desirably carried out at low cost and various test methods have been developed. Besides diagnosis of diseases based on a single gene, testing multiple genes has become important in relation to diseases caused by various genes and environmental effects and to drug sensitivity, in medical diagnosis. Accordingly, it is important to simultaneously examine various kinds of genes. Therefore, it is necessary to examine multiple genes instead of a single gene or mutation and thus there is a need for a system which includes processes for amplification of a region to be tested in a gene of interest and measures SNPs at low cost. For that purpose, a simple method which is different from capillary gel electrophoresis used in genome analysis is needed. Reported examples of the system to be used for SNP analysis or probe tests for genes include an invader assay, Taqman assay, DNA chip, and pyrosequencing (Science 281, 363 (1998)). The former three are detection methods using fluorophore tags, which include an excitatory laser light source and a light detection system. The pyrosequencing is a system using phased complementary strand extension and bio-luminescence, which includes a system for injecting a trace amount of nucleotide substrates in a designated order and a light detection system. Recently, the present inventors have reported a method for detecting SNPs, in which the 3′-terminal region of a primer is hybridized with a SNP site of a target DNA to proceed complementary strand extension and the resulting pyrophosphate is converted into ATP to generate bio-luminescence. In this case, the complementary strand extension characteristically takes place only when the primer is securely hybridized at the 3′ terminus with the target DNA, thereby a wild-type target DNA and a mutant target DNA can be discriminated (Seibutsu Butsuri Kagaku 45, 219–225 (2001)). On the other hand, SNP analysis methods using gel electrophoresis have come into wide use. The simplest is a method in which a single-base complementary strand extension reaction is carried out using four kinds of fluorophore-tagged terminators and the fluorophore-tagged terminators and fluorophore-tagged DNAs are separated and measured using gel electrophoresis (Bio Wave 17, 2–5 (2001)). This method will be explained in detail since it mostly relates to the present invention. Multiple copies of a target DNA are prepared by PCR or the like. Although either double-stranded DNA or single-stranded DNA can be used for the analysis, a single-stranded DNA is used here as a target DNA to simplify the explanation. A primer, the 3′ terminus of which is designed to come before the site to be tested for mutation of the target, is hybridized with the target to proceed single-base complementary strand extension. Here, four kinds of fluorophore-tagged terminators have been added in advance as a substrate for the complementary strand extension reaction. No further complementary strand extension takes place when any one of the nucleotide substrates (in this case, terminators) is incorporated. Only one of the fluorophore-tagged terminators is incorporated into the complementarily extended primer. Multiple fluorophores cannot bind to the primer to proceed complementary strand extension. The length of extended complementary strands thus formed is the same independently of the presence or absence of mutant bases in the target DNA and the kind of tagged fluorophores is different depending on the presence or absence of the mutation. Since a large number of unreacted fluorophore-tagged ddNTPs are present in the reaction solution, the fluorescence cannot be detected in this state. Accordingly, the fluorophore-tagged ddNTPs and extended fluorophore-tagged DNA strands are separated by gel electrophoresis and the kind of mutations (SNPs) is determined using a spectral fluorometer.
In a DNA testing method for medicinal diagnoses, for example, the following conditions are important: (1) costs for apparatus, reagents and the like are low, (2) high speed, high throughput detection is possible, (3) multiple DNA sites can be diagnosed simultaneously, and (4) a single-base mutation can distinguishably be detected. Among various methods, widely used is a method using a single-base complementary strand extension reaction, in which the reaction products are subjected to mass spectrometry or capillary gel electrophoresis analysis. However, a mass spectrometer is expensive and preparation of samples for the measurement is disadvantageously time consuming although the measurement itself can be done in a short time. On the other hand, in a method using capillary gel electrophoresis, the length of migration paths is long and it takes 30–60 minutes for detection since a commertially available DNA sequencer is used. Furthermore, four kinds of terminators (ddNTPs), each tagged with different fluorophores, are used as a substrate for complementary strand extension reaction and thus an expensive measuring instrument having a color differentiating function is required. The above mentioned methods are not necessarily suitable to carry out SNP measurement or high-throughput analysis readily in a short time and accordingly, the development of new technology for SNP analysis is in need.